RNA regulation provides a mechanism to rapidly control gene expression in response to stimuli, including environmental changes. This project seeks to generate and utilize structural information to enhance our understanding of these processes with an emphasis on the importance of RNA target specificity for proper gene regulation. In this fiscal year, we have studied the atomic structures and functions of classical PUF proteins, which are sequence-specific RNA-binding proteins that are important regulators of gene expression for embryonic development and germline stem cell maintenance, and discovered new PUF proteins that function to regulate pre-ribosomal RNA processing in ribosomal biogenesis. Beginning with determining the first crystal structure of a PUF protein in complex with RNA to recent work on the specificity of human, yeast, and C. elegans proteins, we have identified both common and unique features of RNA recognition by this family of proteins. We now extend this work by showing how two RNA-binding proteins work cooperatively to recognize and regulate gene expression. We have determined crystal structures of the PUF protein Pumilio in complex with the zinc-finger protein Nanos and their target RNAs. These structures show how Nanos protein extends the RNA recognition site of the Pumilio-RNA complex, but it also changes the RNA recognition properties of Pumilio. This is the first demonstration of such a phenomenon. What this means is that the activity of two RNA-binding proteins is not merely additive, but can also expand the range of mRNA targets. Given the importance of human Pumilio and Nanos proteins in embryonic development and germline stem cells, these findings will help to understand what RNAs are regulated in these processes. This work was published in eLife. We have also used crystal structures, biochemical assays, and in vivo studies to identify new families of PUF-related proteins that operate in ribosomal biogenesis. We have discovered that the protein Nop9 functions in ribosomal biogenesis. This new PUF-like protein, like the Puf-A/Puf6 protein that we discovered previously, has different RNA recognition properties than the classical PUF proteins. Nop9 binds to a structured stem loop RNA in the pre-ribosomal RNA, and it recognizes both sequence and structural features. By binding to this stem loop in the pre-ribosomal RNA, Nop9 controls the timing of maturation of the ribosomal RNA. This work is in press at Nature Communications. We also used structural and biophysical studies to contribute to understanding the effects of mutations in the ribosomal biogenesis factor Nop4/RBM28. We found that a specific point mutation that causes the human disease alopecia, neurological defects and endocrinopathy (ANE) syndrome disrupts the structure of RBM28 and disrupts Nop4s function in bringing together proteins important for ribosome assembly. This study was published in eLife.